Protein aging hypothesis of Alzheimer disease.

Alzheimer disease (AD), the most common form of aging-related neurodegenerative disorders, is associated with formation of fibrillar deposits of amyloid beta-protein (Abeta). While the direct involvement of Abeta in AD has been well documented, the relations between Abeta production, amyloid formation, and neurodegeneration remain unknown. We propose that AD is initiated by a protein aging-related structural transformation in soluble Abeta. We hypothesize that spontaneous chemical modification of aspartyl residues in Abeta to transient succinimide induces a non-native conformation in a fraction of soluble Abeta, rendering it amyloidogenic and neurotoxic. Conformationally altered Abeta is characterized by increased stability in solution and the presence of a non-native beta-turn that determines folding of Abeta in solution and the structure of Abeta subunits incorporated into amyloid fibrils. While the soluble 'non-native' Abeta is both the factor triggering the neurodegenerative cascade and the precursor of amyloid plaques, these two events result from interaction of Abeta with different sets of cellular components and need not coincide in space and time. Extensive literature data and experimental evidence are provided in support of this hypothesis.

Induction of beta-sheet structure in amyloidogenic peptides by neutralization of aspartate: a model for amyloid nucleation.

Amyloid fibril formation is widely accepted as a critical step in all types of amyloidosis. Amyloid fibrils derived from different amyloidogenic proteins share structural elements including beta-sheet secondary structure and similar tertiary structure. While some amyloidogenic proteins are rich in beta-sheet in their soluble form, others, like Alzheimer beta-amyloid peptide (Abeta) or serum amyloid A, must undergo significant structural transition to acquire a high beta-sheet content. We postulate that Abeta and other amyloidogenic proteins undergo a transition to beta-sheet as a result of aging-related chemical modifications of aspartyl residues to the form of succinimide or isoaspartyl methyl ester. We hypothesize that spontaneous cyclization of aspartate residues in amyloidogenic proteins can serve as a nucleation event in amyloidogenesis. To test this hypothesis, we synthesized a series of designed peptides having the sequence VTVKVXAVKVTV, where X represents aspartic acid or its derivatives. Studies using circular dichroism showed that neutralization of the aspartate residue through the formation of a methyl ester or an amide, or replacement of aspartate with glutamate led to an increased beta-sheet content at neutral and basic pH. A higher content of beta-sheet structure correlated with increased propensity for fibril formation and decreased solubility at neutral pH.

Isoaspartate in chrondroitin sulfate proteoglycans of mammalian brain.

Mammalian brain contains a high mass protein (HMAP) that is unusually rich in atypical L-isoaspartyl (isoAsp) linkages. HMAP has now been purified from bovine brain by anion exchange, hydroxylapatite, and size exclusion chromatography. It is self-aggregating, acidic, and soluble in 5% trichloroacetic acid. Treatment with chondroitinase ABC eliminates the self-aggregation of HMAP and generates several distinct core proteins with estimated masses of 350-450 (doublet), 180, and 100 kDa, indicating that it is composed mainly of chondroitin sulfate proteoglycans (CSPGs). Most of the isoAsp resides in the 350-450-kDa core protein, which was identified by immunoblotting as phosphacan, a CSPG abundant in adult brain. The regional distribution and developmental profile of HMAP in rat brain support this identification. The 180-kDa core protein contains a tenascin-R-related molecule, consistent with recent observations that phosphacan forms a tight complex with tenascin-R. The average phosphacan molecule in adult brain contains at least seven isoAsp sites. Molecular heterogeneity due to isoAsp may explain some of the complex binding properties phosphacan exhibits with its natural ligands. Formation of isoAsp may be important in the roles that phosphacan and other CSPGs play in development of the nervous system.

High mass methyl-accepting protein (HMAP), a highly effective endogenous substrate for protein L-isoaspartyl methyltransferase in mammalian brain.

A previously unidentified endogenous substrate for protein L-isoaspartyl methyltransferase in mammalian brain has been characterized and partially purified. This high mass methyl-accepting protein (HMAP) is concentrated in rat brain cytosol and is not detectable in rat liver, heart, lung, kidney, or skeletal muscle. HMAP is acidic and heterogeneous in size, with an average mass, as judged by size-exclusion high performance liquid chromatography, greater than 700 kDa. After partial purification from cow brain by anion-exchange chromatography, ammonium sulfate fractionation, and gel filtration, HMAP could accept 12.1 nmol of methyl groups per mg of protein, suggesting that it contains a level of isoaspartate at least 50 times greater than that of the average protein in brain cytosol. Partially purified HMAP is degraded by trypsin, verifying that it is composed, at least in part, of protein. Additional studies on this unusual macromolecule may shed important new light on mechanisms of isoaspartate formation in cells and the molecular pathology of brain aging.

Molecular aging of tubulin: accumulation of isoaspartyl sites in vitro and in vivo.

The formation of isoaspartyl sites during aging of rat tubulin in vitro and in vivo has been studied. When incubated in vitro at pH 7.4, 37 degrees C, purified rat brain tubulin accumulated isoaspartyl sites at a rate > or = 2.4 isoaspartyl sites per 100 tubulin subunits (50 kDa) per day for 30 days. Isoaspartate levels were estimated by the transfer of radiolabeled methyl groups from S-adenosyl-L-[methyl-3H]-methionine in a reaction catalyzed by protein-L-isoaspartyl methyltransferase. isoaspartate formation occurred in parallel with, but was not dependent upon, extensive cross-linking of tubulin via formation of intermolecular disulfide bonds. When rat PC12 cells were incubated for 24 or 72 h in the presence of adenosine dialdehyde, a potent methyltransferase inhibitor, a substantial and consistent increase in the isoaspartate content of tubulin was observed. This suggests that tubulin constantly undergoes isoaspartate formation in vivo, but that the levels are normally kept low by methylation-dependent repair. These findings support the hypothesis that protein-isoaspartyl methyltransferase plays a key role in countering spontaneous damage reactions to proteins associated with cell aging. These results also suggest that tubulin is an important target for protein-isoaspartyl methyltransferase in vivo.

Multiple forms of O-methyltransferase involved in the microbial conversion of abietic acid into methyl abietate by Mycobacterium sp.

Six out of seven tested strains of mycobacteria transformed abietic acid to methyl abietate in shake culture. The conversion carried out by Mycobacterium sp. MB 3683 was induced by the substrate and stimulated by methionine. Fractionation of the cell extract of Mycobacterium sp. MB 3683 on DEAE cellulose, Ultrogel AcA 44 and MONO Q resulted in the separation of three distinct methyltransferase activities which could also esterify palmitic acid. The separated forms of the methyltransferase exhibited different activities towards these two substrates.

Novel metabolite structures from biotransformation of a sesquiterpenoid ketone by selected fungal strains.

The sesquiterpenoid ketone, 1,4,4-trimethyltricyclo[5.4.0.0(3.5)]undec-7-en-9-one (1), was subjected to microbial transformation by six fungal strains: Aspergillus niger ATCC 9142, Aspergillus ochraceus DSM 824, Beauveria bassiana ATCC 7159, Cunninghamella echinulata ATCC 9244, Rhizopus arrhizus ATCC 11.145, and Absidia blakesleeana ATCC 10.148. Four main metabolites were formed from 1: 10(R)- and 10(S)-hydroxy-1,4,4-trimethyltricyclo-[5.4.0.0(3.5)]undec-7- en- 9-one (2 and 3, respectively), besides 4(R)- and 4(S)-(hydroxymethyl)-1,4-dimethyltricyclo[5.4.0.0(3.5)]undec -7-en-9-one (4 and 5, respectively). Compounds 2-5 were isolated with varying percentages from the respective transformations, and their structures established unequivocally by a combination of spectroscopic methods. Metabolites 2 and 3 are products of hydroxylation at C-10, in either R- or S-position; in 4 and 5, one geminal CH3 group each on the cyclopropane ring has been transformed into a CH2OH function.